1. Field of the Invention
This invention relates to an analytical element for immunoassay utilizing antigen-antibody reaction and preparation thereof.
2. Description of the Prior Art
Recently, analytical methods using dry-type analytical elements have been developed. In a dry-type analytical element, generally, an analyte, such as a biochemical substance contained in a body fluid, is allowed to react with reagent(s) incorporated in the analytical element. The amount of a particular reaction product or unreacted component is then determined by measuring the coloring, discoloring, fluorescence, luminescence or the like, optically, such as by spectrophotometry, to determine the content of the analyte. By using the dry-type analytical element, a particular component, such as a biochemically active substance, in a liquid sample can be analyzed simply, rapidly and acurately.
On the other hand, an immunoassay utilizing a dry-type analytical element has been reported in U.S. Pat. Nos. 3,992,158 and 4,459,358, German Patent Application 3 343 695A.
The antigen-antibody reaction in the field of immunology is the reaction in which an antibody or antigen specifically reacts with the corresponding antigen or antibody alone. It is widely utilized for the diagnosis of diseases of autoimmune system, detection of a trace component in a living body and the like. However, since methods utilizing antigen-antibody reactions require significant operational skills, it has been desired in the field of clinical tests to develop a measuring method requiring only simple operations which produces accurate results.
The method of measuring an antigen disclosed in EP 979 52A is a typical method utilizing a multilayer analytical element. This analytical element is composed of a spreading layer containing an antigen labeled with a fluorescent material, a partition layer composed of a porous medium and a reaction layer containing an immobilized antibody. The amount of the antigen is determined in this element by measuring the decrease of fluorescence intensity, utilizing the competitive antigen-antibody reactions between the antigen in a sample solution and the labeled antigen.
As another immunoassay method having a relatively high sensitivity, enzyme immunoassay (EIA) is known. A typical EIA is the solid phase competitive reaction method comprising allowing the antigen to be measured and an enzyme-labeled antigen or its analogue to react competitively with immobilized antibody, conducting bound-free (B/F) separation, and measuring the activity of either the enzyme bound to the antibody or the free enzyme to determine the amount of the antigen to be measured. In order to eliminate the B/F separation, an enzyme whose activity increases or decreases by binding to the antibody is necessary.
In order for these measuring systems to work effectively, two components, i.e. an enzyme-labeled antigen or its derivative and an immobilized antibody in one case, or an enzyme labeled antibody or its derivative and an immobilzed antigen in the other case, are necessary. In either case, these two components should be isolated so as not to react with each other before measuring. Satisfactory analytical sensitivity was not obtained in most of the known dry-type analytical elements because of a low signal/background ratio caused by insufficient isolation of the two components.